KR101928543B1 - Pharmaceutical Composition for Treating or Preventing Disease Concerining Axin-GSK3 Binding Containing Niclosamide - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prophylaxis of Axin-GSK3 protein binding-related diseases such as familial adenomatosis polyposis (FAP) containing nickelrosamide or a pharmaceutically acceptable salt thereof as an active ingredient According to the present invention, using FDA-approved safe drug, nickelosamide, can effectively treat familial adenomatosis polyposis (FAP) suffering from no treatment.

Description

[0001] The present invention relates to a pharmaceutical composition for treating Axin-GSK3 protein binding-related diseases containing nickel,

The present invention relates to a pharmaceutical composition for treating Axin-GSK3 protein binding-related diseases containing nickel chloride, and more particularly to a pharmaceutical composition for treating Axin-GSK3 protein binding-related disease, which comprises nickellazamide or a pharmaceutically acceptable salt thereof as an active ingredient, , familial adenomatosis polyposis), multiple colonic polyps, inflammation caused by Helicobacter pylori, or gastric cancer.

The epithelial-mesenchymal transition (EMT) is a biological mechanism that induces invasive, stem cell-mediated resistance to treatment in human cancer, and a method of treating cancer by returning EMT as a low-molecular inhibitor has been studied . Salinomycin is known to effectively reverse the EMT of cancer cells, and has been reported to inhibit cancer cell differentiation up to 100-fold in proportion to paclitaxel, a representative anticancer drug, and inhibit metastasis of breast cancer in vivo experiments (Gupta PB, et al. , Cell ., 138: 645-6592, 2009). Although salinomycin is not available to humans, these results suggest new cancer treatment strategies that control EMT in human cancers.

The transcription factor Snail induces EMT by inhibiting the genes of epithelial tissue in human cancers (Cano A. et al., Nature Cell Biol, 2, 76-83, 2000; Batlle E et al., Nature Cell Biol, 84-89,2000). The major carcinogenic pathway of the Wnt signal, the p53 tumor suppressor gene and the H. pylori bacterial carcinogen CagA protein regulates Snail activity through post-translational, post-transcriptional mechanisms (Yook JI et al., J Biol Chem, 280, 11740 Kim et al., J Cell Biol 195, 417-433, 2011; Lee et al., Nature Communications, 5: 4423, 2014). In addition, the β-catenin and Snail transcription mechanisms are phosphorylated and degraded by GSK3. In addition, the Wnt signal or CagA inhibits phosphorylation, thereby increasing the TCF transcriptional activity and promoting Snail-mediated EMT. Axin2, a GSK3 scaffolding protein, functions to regulate this process by regulating nuclear-cytoplasmic shuttling of GSK3 (Yook JI et al., Nature Cell Biol, 8, 1398-1406, 2006). As a result, an increased nucleus of cancer occurs in cancer cells. Interestingly, the GSK3 shuttle ring function by Axin is required for phosphorylation of the membrane LRP6 Wnt receptor and subsequent activation of intracellular Wnt signaling. Thus, the Axin-GSK3 complex plays an important role in regulating the Wnt signal and the Snail-mediated EMT program. Conversely, inhibition of the Axin-GSK3 complex can be a new mode of action (MoA) in the development of low molecular inhibitors for the Wnt signaling of human cancers and the Snail-mediated EMT program.

Helicobacter pylori (Helicobacter pylori) is a typical cancer-causing factor living in a person's stomach. The World Health Organization (WHO) defines Helicobacter as a definite carcinogen bacterial infection. Helicobacter infections occur in more than 50% of the population in Korea (Leomardo et al., Helicobacter, 19, supple 1: 1-5, 2014), with a prevalence of 30-40% in the West and 70% in the underdeveloped countries. Helicobacter is infected with human gastrointestinal tract, causing inflammation of gastric mucosa, and furthermore, it is an important cause of gastric cancer. Helicobacter is infected with gastrointestinal tract and injects cytotoxin-associated gene A (CagA) protein into gastric mucosal cells, which plays an important role in gastrointestinal inflammation and cancer development. It has recently been found that CagA performs similar functions to Axin. Specifically, when CagA is injected into epithelial cells of Helicobacter, CagA binds to GSK-3 like Axin, which inhibits GSK-3 phosphorylase function and further increases Snail expression (Lee et al., Nature Communications , 5: 4423, 2014). Although early studies have been reported to increase expression of Snail in cancer metastasis, recent results indicate that Snail directly induces inflammation of epithelial cells as well as cancer development (Lyons et al., Cancer Res. 68; 4525 -4530, 2008; Du et al., Cancer Res. 70: 10080-10089, 2010). Therefore, a compound that binds to GSK-3 and inhibits Axin function and further inhibits Snail expression may be useful for inhibiting Helicobacter-induced inflammation and gastric cancer.

On the other hand, niclosamide is an FDA approved insecticide and has been widely used for infestation of intestinal tapeworms for nearly 50 years. Since nickel dichloride has been reported to be an effective substance of human colon cancer in vivo or in vitro experiments (Osada T, et al ., Cancer Res . 71: 4172-4182, 2011; 13. Sack U, et al ., J Natl Cancer Inst . 103: 1018-1036, 2011), recent studies have reported that nickel chloride can be used in various types of human cancers (Li Y, Li PK, Roberts MJ, Cancer Lett ., 349: 8-14 , 2014; Wang YC et al ., PLoS One . 8: e74538, 2013).

Nucleoside induces cancer cell death at micromolar concentration levels in vitro. On the other hand, physiological concentrations in vivo show nM levels in plasma and cancer tissues and MoA (mode of action) that is not cytotoxic in vivo. While many targets have been proposed, such as Notch signals, Disheveled, S100A4, and Frizzled receptors, the direct target of providing the MoA (mechanism of action) of nickel chloride has not yet been elucidated.

In the present invention, in order to find out a method for treating Axin-GSK3 protein binding-related diseases such as familial adenomatosis polyposis (FAP) and Helicobacter infection, adenoma formation in an APC-MIN animal model of oral administration of nickel- And the present invention was completed.

It is an object of the present invention to provide a pharmaceutical composition for treating Axin-GSK3 protein binding-related diseases.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating or preventing Axin-GSK3 protein binding-related diseases, which comprises nickelclodurate or a pharmaceutically acceptable salt thereof as an active ingredient.

According to the present invention, the FDA-approved safe drug, nickelosamide, can effectively treat familial adenomatosis polyposis (FAP), multiple colonic polyps, and Helicobacter pylori infection, which are suffering from no treatment.

FIG. 1 shows the results of confirming the effect of treatment with nickel chloride on colon cancer cells. FIG. 1 shows the survival rate of colon cancer cells according to the concentration of nickel chloride, B shows beta-catenin expression ) And TCF / LEF transcription activity (right).
Fig. 2 shows changes in Snail expression (A) and E-cadherin activity (B) according to treatment with nickel chloride.
FIG. 3 shows the results of analysis of the amount of GSK3, β-catenin, and Snail in the nucleus-cytoplasmic fraction of colon cancer cells treated with nickel chloride.
Fig. 4 shows the results of confirming that the formation of Axin-GSK3 complex is inhibited by the nicklozamide.
FIG. 5A shows the result of surface plasmon resonance (SPR) analysis to confirm whether nickel chloride binds directly to GSK3, and FIG. 5C shows the results of structural analysis of the Axin binding portion and the nickel concentration of GSK3.
FIG. 6 is a graph showing changes in the cell migration ability (A) of colon cancer cells treated with nickel chloride and the activity of nickel chloride in mice administered with colon cancer cells to confirm epithelial-mesenchymal transition (EMT) restoration ability of nickel chloride (B). ≪ / RTI >
FIG. 7 shows the results of immunoblot assay for changes in the amounts of Snail and E-cadherin proteins by the treatment with nicklozamide in a heterogeneous tumor transplantation experiment.
FIG. 8 shows the results of confirming the change in TCF / LEF transcriptional activity by the nucleoside in 293 cells transformed with the mutant APC.
FIG. 9 shows the results of confirming the change in size of adenomas when nitric oxide was administered to an APC-MIN animal model.
Fig. 10 shows the results of inhibiting the binding between Helicobacter CagA and GSK-3 by nickel chloride and inhibiting Snail expression.

In the present invention, it was confirmed that nickel chloride directly inhibits Axin-GSK3 complex formation in cells, restores Snail-mediated epithelial-mesenchymal transition (EMT) in colorectal cancer cells, and encompasses Wnt activity. In addition, it was confirmed that the inhibition of the activation of the transcriptional activity of TCF / LEF induced by the APC mutation in FAP patients was inhibited by nickel. In addition, it was confirmed that adenoma formation was inhibited when orally administered to the APC-MIN model established to form colon adenoma.

Accordingly, the present invention relates to a pharmaceutical composition for the treatment or prevention of Axin-GSK3 protein binding-related diseases containing nickelrosamide or a pharmaceutically acceptable salt thereof as an active ingredient.

Niclosamide (or Niclocide (TM)) is a drug that has been used as a pesticide for nearly 50 years and is known to have potentially antitumor activity and is an orally available drug.

Nickelosamine has recently been shown to function as an anticancer drug that regulates many signaling pathways, such as Wnt, S100A4, Notch, and androgen receptors, although the molecular target has not been clearly elucidated (Osada T, et al. Cancer Res Li, Y. et al., Cancer Lett ., 349: 8-14, 2014 (2001), pp. 41-4172-4182, 2011; Sacki, et al. J Natl Cancer Inst . 2011; 103 (13): 1018-1036 ).

FAP (familial adenomatosis polyposis) is caused by a deficiency of APC (adenomatous polyposis coli), and FAP patients develop hundreds of adenomatous polyps in the intestines at an early age and ultimately 100% colorectal (Minde DP et al., Mol Cancer . 2011; 10: 101, 2011; Half E et al. , Orphanet J Rare Dis ., 4: 22, 2009). The US FDA and the European Health Organization have approved anti-inflammatory drugs with some additional treatments, but the therapeutic effect is minimal.

Oral administration of the nickel rosamide according to the present invention may be effective as a new drug for treating FAP.

In one embodiment of the present invention, the administration of the nicotinamide to the APC-Min mouse model with the same genetic background and symptoms as the FAP patient resulted in significantly reduced intestinal adenoma at 14 weeks for rats injected with niclosamide into the peritoneum . On the other hand, his weight did not change. Intraperitoneal adenoma formation was significantly inhibited in the APC-MIN model of oral administration of niclosamide for 14 weeks, and the experimental animals were stable during drug treatment.

These results indicate that nickel chloride can be used as a new therapeutic agent for FAP patients.

In addition, the present invention shows the results of inhibiting inflammation and gastric cancer progression caused by Helicobacter infection, and these results can be effectively used for various inflammatory diseases caused by reduction of GSK-3 activity.

In the present invention, it was confirmed that nickel chloride inhibited the binding of Axin and GSK3 to form a complex to weaken Wnt activity and restore the Snail-mediated EMT in colon cancer cells. It also provided clinical validity of nickel chloride as a potential treatment for FAP patients.

In one embodiment of the present invention, it was confirmed whether or not nicklozamide induces cell death of colon cancer cells. As shown in Fig. 1 A, it was confirmed that nickelosamide induces the death of colon cancer cells at the concentration of μM , Whereas cell death was not observed at the concentration of nM.

In the present invention, it was confirmed that nickeloside was used at the concentration of nM to inhibit colon adenoma by a mechanism other than cancer cell death.

In the present invention, GSK3-Axin2 interaction was confirmed as a molecular target of nickelosamide. Nickelosamide has restored Snail-mediated EMT in cancer cells and provided new MoA that inhibits Wnt activity.

Accordingly, the present invention provides a method for treating or preventing Axin-GSK3 protein binding-related diseases, wherein the Axin-GSk protein binding-related disease is FAP (familial adenomatosis polyposis), adenomatous polyposis, Diabetic rheumatoid arthritis, inflammatory skin disease, osteoarthritis, leukopenia, and the like.

In the present invention, the abnormality of the Wnt signal pathway is also associated with many diseases including cancer, bone metabolism, degenerative diseases, fibrosis, and despite the target of Wnt therapy such as Frizzled receptor, Porcupine, Disheveled, p300 and CBP, Effective molecular targets for regulating the Wnt pathway are not yet known.

In the present invention, during Wnt activation, the APC-Axin-Disheveled scaffolding complex modulates TCF / LEF activity. Inhibition of Axin-GSK3 interaction could be a new target to attenuate Wnt activity and restore the Snail-mediated EMT program.

The carrier used in the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier, adjuvant and vehicle and is collectively referred to as a " pharmaceutically acceptable carrier ". Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of the present invention include, but are not limited to, ion exchange, alumina, aluminum stearate, lecithin, serum proteins (e.g., human serum albumin), buffer substances Water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride and zinc salts), colloidal silicon dioxide Polyethylene terephthalate, silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substrate, polyethylene glycol, sodium carboxymethylcellulose, polyarylate, wax, polyethylene-polyoxypropylene-barrier polymer, polyethylene glycol and wool.

The route of administration of the pharmaceutical composition according to the present invention may be, but is not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, Sublingual or rectal.

Oral and parenteral administration is preferred. The term "parenteral" as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

The pharmaceutical composition may be in the form of a sterile injectable preparation, either as a sterile injectable aqueous or oleagenous suspension. The suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents (e. G., Tween 80) and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent (e.g., a solution in 1,3-butanediol). Vehicles and solvents that may be used tolerably include mannitol, water, a Ringgel solution and an isotonic sodium chloride solution. In addition, sterile, nonvolatile oils are conventionally employed as a solvent or suspending medium. For this purpose, any non-volatile oil with low irritation, including synthetic mono- or diglycerides, may be used. Fatty acids such as oleic acid and glyceride derivatives thereof are useful in injection formulations as well as pharmaceutically acceptable natural oils (e.g., olive oil or castor oil), especially those polyoxyethylated.

The pharmaceutical compositions of the present invention may be orally administered in any dosage form orally acceptable, including, but not limited to, capsules, tablets, and aqueous suspensions and solutions. In the case of oral tablets, commonly used carriers include lactose and corn starch. Lubricants such as magnesium stearate are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When the aqueous suspension is orally administered, the active ingredient is combined with an emulsifying agent and a suspending agent. If desired, sweetening and / or flavoring agents and / or coloring agents may be added.

The pharmaceutical compositions of the present invention may also be administered in the form of suppositories for rectal administration. These compositions may be prepared by mixing the compounds of the invention with suitable non-polar excipients which are solid at room temperature but liquid at rectal temperature. Such materials include, but are not limited to, cocoa butter, beeswax, and polyethylene glycols.

Oral administration of a pharmaceutical composition according to the present invention is particularly useful when the desired treatment is associated with a site or organ that is accessible by topical application. When topically applied to the skin, the pharmaceutical composition should be formulated with a suitable ointment containing the active ingredient suspended or dissolved in the carrier. Carriers for topical administration of the compounds of the present invention include, but are not limited to, mineral oil, liquid paraffin, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax and water. Alternatively, the pharmaceutical composition may be formulated with a suitable lotion or cream containing the active compound suspended or dissolved in a carrier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. The pharmaceutical compositions of the present invention may also be topically applied by rectal suppositories to the lower intestinal tract with a suitable enema. Topically applied transdermal patches are also included in the present invention.

The pharmaceutical composition of the present invention can be administered by inhalation aerosol or by inhalation. Such compositions may be prepared according to techniques well known in the art of pharmacy and may be prepared using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other solubilizing or dispersing agents known in the art, Solution.

The compounds of the present invention can be used in combination with conventional anti-inflammatory agents or in combination with inhibitors of matrix metalloproteinase inhibitors, lipoxygenase inhibitors and cytokines other than IL-1beta. The compounds of the present invention may also be used in combination with immunomodulators (e.g., bropyrimines, anti-human alpha interferon antibodies, IL-2, GM-CSF, methionine enkephalin, interferon Alpha, diethyldithiocarbamate, tumor necrosis factor, naltrexone and rEPO) or prostaglandins. When the compounds of the present invention are administered in combination with other therapeutic agents, they may be administered to a patient sequentially or simultaneously.

The term " therapeutically effective amount " refers to a dosage level (typically from about 60 mg to about 6 g / patient / day) of about 1 mg to about 100 mg per kg body weight per day for use in the treatment of the condition in humans.

The term " prophylactically effective amount " refers to a dosage level (typically from about 6 mg to about 6 g / patient / day) of about 0.1 mg to about 100 mg per kg body weight per day for use in the prevention of the condition in humans.

It will be understood, however, that the specific effective amount for a particular patient will depend upon a variety of factors, including the activity of the particular compound employed, age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and severity of the particular disease It will be appreciated that it may vary depending on factors. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.

When the medicinal composition according to the present invention is injected into subcutaneous cells of a fish, it can be administered to a dacryocyst or digestive tract. Injection can be injected into muscle cells or other cells within the muscle tissue and injected into the intestinal cells in the abdominal cavity.

In a preferred embodiment, the pharmaceutical compositions for oral administration can be prepared by mixing the active ingredients together with solid excipients and may be prepared in the form of granules for preparation in the form of tablets or dragees. Suitable excipients include sugars such as lactose, sucrose, mannitol and sorbitol or starches from corn, wheat flour, rice, potato or other plants, cellulose such as methylcellulose, hydrocyclopropylmethylcellulose or sodium carboxymethylcellulose , Arabic gum, carbohydrates such as gum including Tagakans gum, or protein fillers such as gelatin and collagen. If necessary, disintegrating or solubilizing agents in the form of respective salts such as cross-linked polyvinylpyrrolidone, agar and alginic acid or sodium alginic acid may be added.

In a preferred embodiment, for parenteral administration, the pharmaceutical composition of the present invention can be prepared as a water-soluble solution. Preferably, physically suitable buffer solutions such as Hank's solution, Ringer's solution or physically buffered saline can be used. Water-soluble injection suspensions may contain a substrate capable of increasing the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. In addition, suspensions of the active ingredient may be prepared in suitable oily injection suspensions. Suitable lipophilic solvents or carriers include fatty acids such as sesame oil or synthetic fatty acid esters such as ethyl oleate, triglycerides or liposomes. Polycationic amino polymers can also be used as carriers. Optionally, the suspension may employ a suitable stabilizing agent or agent to increase the solubility of the compound and to produce a high concentration of solution.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1: Analysis of cytotoxic activity of colorectal cancer cells by nickel

In order to confirm whether nickelosamide induces apoptosis of colon cancer cells, the effect on survival rate and mobility was confirmed.

1 × 10 ⁵ cells of the colon cancer cell line HCT116, SW480 and DLD-1 cells (ATCC, American Type Culture Collection) were cultured overnight in a 6-well plate, washed with PBS, 0.125 μM, 0.25 μM, 0.5 μM, 1 μM, 2 μM, 5 μM and 10 μM) for 48 hours in a culture medium. Cell death was measured with a trypan blue assay, and cell viability was calculated by the equation [1- (number of dead cells / total number of cells)].

As a result, as shown in Fig. 1 (A), it was confirmed that nickelosamine induces the death of colon cancer cells at the concentration of μM, while cell death was not observed at the concentration of nM.

Example 2: Confirmation of snail-mediated EMT reversion activity of colorectal cancer cells by nickel

To confirm that niclosaramide induces snail-mediated EMT reversion, β-catenin expression, TCF / LEF reporter (Topflash) activity changes, Snail and E-cadherin protein expression were confirmed.

First, colorectal cancer cells were treated with nitrosamides of nM units at concentrations of 0 nM, 0.125 nM, 0.25 nM and 0.5 nM for 24 hours, and then the β-catenin activity and the TCF / LEF reporter (Topflash) activity change was measured.

Β-catenin was Western blotted using β-catenin (# 610154, BD Transduction, 1: 5,000) antibody. As a result, as shown in FIG. 1B (left) The expression of catechin decreased with increasing treatment concentration of nickel.

TCF / LEF transcriptional activity was transformed with 100 ng of reporter gene and 1 ng of transfection control pRL-SV40-Renilla. Reporter activity was measured 48 hours after infection by the dual luciferase assay system (Promega) and standardized by measuring the co-transformed renilla activity, and the reporter gene activity was expressed in light units proportional to the light units obtained from the negative control.

As a result, as shown in B (right side) in Fig. 1, the TCF / LEF transcription activity decreased as the treatment concentration of the nickel rosamide increased.

Western blot analysis confirmed the expression of Snail and the expression of E-cadherin protein was confirmed through a dual luciferase assay system. As a result, as shown in FIG. 2A, as the treatment concentration of nickel , While E-cadherin increased, as shown in Figure 2B.

It was judged that the Snail functioning as an E-cadherin transcription inhibitor was decreased by the treatment with the nucleoside and the E-cadherin promoter activity was increased in the colorectal cancer cells.

Example 3: Confirmation of axin2 function in nicotinamide colon cancer cells

To confirm whether or not the inhibition of Axin2 function in colorectal cancer cells, the change of GSK3 level in the cell nucleus was observed.

Colorectal cancer cell lines HCT116, SW480 and DLD-1 cells were treated with 0.25 nM of niclosaramide for 24 hours, and the amount of GSK3, β-catenin and Snail in the nuclear-cytoplasmic fraction was analyzed by immunoblotting.

The amount of Snail and GSK3 protein in the cells was determined by separating the nucleus and cytoplasm from the stock solution (Kim, NH, et al ., Sci . Signal 4: ra71, 2011; Yook JI, et al ., Nat Cell Biol ., 8: 1398 -140, 2006). Briefly, colon cancer cells (1 x ¹⁰⁶ cells) were collected in centrifuge tubes, washed with PBS and resuspended in ice-cold buffer solution (10 mM HEPES, pH 7.9; 10 mM KCl; 1 mM DTT with protease inhibitors). The cell membrane was further destroyed with 10% NP-40, adjusted to a final concentration of 0.6%, mixed well and centrifuged at high speed for 30 seconds. The cytoplasmic degradation was separated from the supernatant and the nuclear precipitate was washed twice with cold PBS. The nucleoprotein was extracted by treating with ice-water buffer (20 mM HEPES, pH 7.9; 0.4 M NaCl; 1 mM DTT with protease inhibitors) on ice for 15 minutes and then centrifuged at high speed and GSK3, β-catenin And Snail were immunoblotted and analyzed.

As a result, as shown in Fig. 3, nucleoside GSK3 was increased by the treatment with the nucleoside, and the amounts of beta-catenin and snail were decreased.

The above results indicate that nickelosamide can modulate the function of Axin in colorectal cancer cells.

Example 4: Confirmation of inhibition of axin-GSK3 complex formation of nickelosamide

In order to confirm whether nickelosamides can inhibit the Axin-GSK3 interaction, cell lysates with full-length Axin2 were investigated in order to perform immunoprecipitation experiments with and without nickelosamides.

Immunoprecipitation assays were performed as follows (Yook JI, et al. Nat Cell Biol . 8: 1398-1406, 2006).

The isoxycycline-induced His-tagged Axin2 expression vector was transformed with MCF-7 cells (ATCC, American Type Culture Collection) and cultured. To the Triton X-100 lysate of all cells, Ni-Ti beads (Invitrogen) Lt; RTI ID = 0.0 > of < / RTI > Proteins recovered in the beads were subjected to SDS-PAGE, followed by immunoblot analysis for GSK3, and a 1/20 volume control was added.

As a result, it was confirmed that, as shown in FIG. 4A, it decreased GSK3 binding to Axin2 in the whole cell culture solution.

Structural analysis of the previously reported Axin- GSK3 binding revealed that the hydrophobic residues of the alpha helices of Axin are deposited in a hydrophobic groove formed by the C-terminal loop of GSK3 (Dajani R et al. , EMBO J. 22: 494 -501, 2003), we hypothesized that nickelosamides would bind to the hydrophobic groove of GSK3 and interfere with the function of Axin. To confirm this, we used recombinant GSK3 binding to 19-mer FITC-conjugated Axin peptide In vitro assays were designed to identify competitive inhibition by niclosamide.

His-tagged recombinant GSK3 beta was obtained from sf9 insect cells in a known manner (Lee DG, et al. Nat Commun . 5: 4423, 2014). FITC-linked 19-mer Axin peptide (Axin1, 383-401, VEPQKFAEELIHRLEAVQR), known to bind to GSK3 as an amphipathic alpha -helix, was chemically synthesized (Peptron). His-tagged recombinant GSK3 and Ni-Ti beads were treated with nickel chloride for 2 h, washed 3 times with PBS, and then incubated with Ni- Quantitative fluorescence measurements were performed using Ti beads. The fluorescence intensity was proportional to the fluorescence intensity obtained from the negative control from the third experiment.

As a result, it was confirmed that the interaction between the recombinant GSK3 and the synthesized Axin peptide was inhibited in proportion to the administration concentration of nickelosamide (Fig. 4B).

Surface plasmon resonance (SPR) analysis was performed to determine if nickel chloride directly binds to GSK3.

SPR was purified using ProteOnTM XPR36 Protein Interaction Array system (Bio-Rad Laboratories, Inc., CA, USA) and the purified recombinant GSK3 beta was immobilized on ProteOn GLH sensor chip. (VEPQKAAEEAIHRAEAVQR, mutation underlined) was diluted with phosphate-buffered saline + Tween 20 + 1% DMSO at different concentrations and then flowed to the chip at a rate of 100 μl / min The results were analyzed by Proteon Manager Software 2.0 using the standard Langmuir models for fitting kinetic data. The complex formation rate is expressed by the coupling constant (ka, in the unit of M-1s-1), and the composite attenuation rate is expressed by the dissociation constant (kd, in the unit of s-1)

       ka

A + B? AB (1)

      kd

The high affinity interaction appears as a low dissociation constant and the rapid "on rate" or "high ka" and the "slow" off rate (or low kd) kd / ka.

As a result, as shown in Figures 5A and B, the wild-type Axin peptide or the nickeloside directly bound to GSK3. The equilibrium dissociation constant K _D is the respective value from SPR analysis of 35 μM, 34 μM. Mutated Axin peptides with mutations in the hydrophobic residues immobilized on the GSK3 protein on the sensor surface failed to bind up to 100 micromolar.

In addition, a molecular docking assay was performed to structurally analyze the interaction of the Axin binding portion of GSK3 with the nucleoside.

Molecular docking measurements were performed using the Maestro 10.4 molecular docking suite. The crystal structure of the (pTyr216) -GSK3? With the Axin peptide was measured by RCSB Protein Data Bank (PDB ID: 3ZDI). All water molecules and metal ions were removed, hydrogen atoms were added to the protein, and an Epik module was used to add both to the sample and other ligands at physiological pH. All compounds were minimized in energy using LigPrep and docked to the receptor structure using the standard precision (SP) module of the Glide Docking Module within the Schrodinger Suite. Prior to the Glide docking analysis, a receptor grid box was created at the center of the co-crystal ligand. Post-minimization was performed to optimize the geometric shape.

As a result, as shown in Fig. 5C, the 1-chloro-3-nitrobenzene group of nickel chloride enters the hydrophobic pores formed from Val 263, Leu 266, Val 267 and Ile 270 residues of human GSK3 beta, p < 293 > Nicklosamide additionally forms a hydrogen bond with Pro294, Thr275, Val 263, and a halogen bond with Tyr288 on the surface of Axin-GSK3. Thus, it has been shown that nickelosamine interferes with the Axin-GSK3 complex by inhibiting protein-protein interactions (PPI).

Example 5: Confirmation of epithelial-mesenchymal transition (EMT) restoration ability of nickelosamide

Snail-induced epithelial-mesenchymal transition (EMT) increases the potential for cell migration and tumor formation. To confirm the ability of nitrosamide to restore epithelial - mesenchymal transition (EMT), the effect of nitrosamide on colon cancer cell migration and tumor formation of colorectal cancer cells was examined.

First, the effect of nitrosamide on the cell migration ability of colon cancer cells was confirmed. Colon cancer cell lines HCT116, SW480 and DLD-1 cells Under the condition of nitrosamide, after 48 hours incubation, the upper side of the membrane was rubbed with a cotton swab and the number of cell movements inserted into the basement membrane was stained with 0.25% crystal violet and counted. Cells were counted in five random fields.

As a result, the migration potential of colorectal cancer cells was greatly reduced when the niclosaramide was treated at the nM level (Fig. 6A).

Next, the effects of the tumorigenicity potential of nickelosamides were investigated in vivo in HCT116, SW480 cells.

Colorectal cancer cell line HCT116 (5 x 10 ⁶ cells) and SW480 (5 x 10 ⁶ cells) were mixed in 100 μl PBS and injected into subcutaneous tissues in female athymic nude mice (6 weeks old). Rats were randomly assigned to two groups and infused intraperitoneally daily with vehicle and niclosaramide for 24 h. Nickeloside was dissolved in 10% Cremophor EL (BASF) and 0.9% NaCl, followed by intraperitoneal injection. After injection of colon cancer cells, rats were observed daily and weight change was measured twice per week. And were measured with calipers when the tumor was observed. Tumor size was calculated by the equation (LXW2) / 2. Where L is the long diameter of the tumor and W is the short diameter.

As a result, as shown in Fig. 6B, intraperitoneal administration of nickel chloride significantly inhibited tumor growth of colon cancer cells in an in vivo experiment (Fig. 6B).

In order to investigate the in vivo MoA of nickel-containing EMT-regulated nitrosamides, changes in the amount of Snail and E-cadherin in the tumor by nitrosamide treatment were observed through xenografts .

Nude mice were subcutaneously injected with SW480 cells (5 x 10 < ⁶ >), a colon cancer cell line. When tumors averaged 500 mm ³ in size, the rats were randomly divided into three groups and injected with vehicle or niclosamide (50 mg / kg, 200 mg / kg) for 3 days in the abdominal cavity. After sacrifice, the tumor tissue portion was separated using Pro-prep protein extraction solution (# 17081, Intron), and the amount of protein of Snail and E-cadherin in the tumor sample was measured by immunoblot assay .

As a result, as shown in Fig. 7, the presence of Snail in the in vivo experimental sample was reduced, while the amount of E-cadherin increased together with increasing the treatment concentration of the nickel rosamide.

From these results, it can be seen that nickelosamides inhibit tumorigenic potential by restoring Snail-mage EMT.

Example 6: Confirmation of inhibition of adenoma formation by nicklozamide

The efficacy of niclosaimide in the treatment of adenomatous colorectal cancer and familial adenomatous polyposis (FAP) caused by mutations in the APC gene has been confirmed.

Based on the results of Examples 2 to 5, in which nickel chloride inhibits Wnt activity and EMT through inhibition of Axin-GSK3, it was confirmed that nickelosamidase could attenuate TCF / LEF transcription activity induced by mutant APC .

293 cells (ATCC) were transfected with the mutant APC expression vector pCMV-neo-Bam APC 1-1309 (# 16508, Addgene) or pCMV-neo-BamAPC 1-1941 (# 16510, Addgene) and Topflash reporter vector , Nicolosamide (0 μM, 0.125 μM, 0.25 μM and 0.5 μM) for 24 hours. The TCF / LEF transcriptional activity was quantitated by measuring luciferase activity in the same manner as in Example 2.

As a result, as shown in FIG. 8, when 293 cells were transfected with the mutant APC, the transcription activity of TCF / LEF was increased, and the transcription activity increased with increasing dose-concentration of the nucleoside.

In addition, in order to confirm the therapeutic effect of the nitrosamide in vivo, the effect of the nitrosamide on the adenoma formation in the APC-MIN (multiple intestinal neoplasia, APCΔ850) mice model was confirmed.

APC-MIN mice were mated with wild-type C57BL / 6J (APC + / +) female and MIN C57BL / 6J (APCMin / +) male. APC-MIN progeny were identified by PCR- I was randomly assigned. (50 mg / kg) was injected intraperitoneally daily (6 days / week), rats were observed daily, and weighed twice weekly. At the end of 14 weeks, mice were sacrificed, whole intestines were obtained, intestinal flakes were opened longitudinally with scissors, washed and unfolded with saline, and tissues were obtained. Tissues were fixed with 10% formalin for 24 hours and washed with 70% alcohol. The fixed intestinal tissues were observed using a stereoscopic microscope, and the size of the adenomas (small, <1 mm; medium 1 to 3 mm; large> 3 mm) was counted for each rat. For oral dosing, APC-MIN rats were fed a daily dose of niclosaramide (15% sugar gel vehicle) (6 days / week) for 14 weeks. The number and size of adenomas were measured by stereoscopic microscopy.

The results are shown in Fig. At 14 weeks, the intestinal adenoma was significantly reduced in rats administered intraperitoneally with niclosaramide. On the other hand, his weight did not change. Intraperitoneal adenoma formation was significantly inhibited in the APC-MIN model of oral administration of niclosamide for 14 weeks, and the experimental animals were stable during drug treatment.

These results indicate that nickel chloride can be used as a new therapeutic agent for FAP patients.

Example  7: Nicklosamide  by Helicobacter  Inhibit

The results of Examples 2 to 5 in which nickel chloride inhibits Wnt activity and EMT through inhibition of Axin-GSK3 and that CagA of Helicobacter pyroli binds to GSK-3 similarly to Axin, And CagA induced Helicobacter pyroli induced GSK-3 binding and Snail induction. When various amounts of nickeloside were added to this sample and examined by immunoprecipitation, it was confirmed that the binding of CagA and GSK3 was inhibited by nicklozamide similar to Axin2 (FIG. 10A). In addition, CagA and Snail expression in 293 cells, followed by treatment with niclosaramide inhibited CagA-induced Snail expression (Fig. 10B). These results indicate that Cyclin A And inhibits the onset of gastric cancer and inflammation caused by Helicobacter.

These results indicate that nickel chloride can be used as a new therapeutic agent for patients infected with Helicobacter pylori.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (3)

Treatment of an Axin-GSK3 protein binding-related disease selected from the group consisting of familial adenomatosis polyposis (FAP) and gastritis due to Helicobacter infection, comprising as an active ingredient nickel dichloride or a pharmaceutically acceptable salt thereof, &Lt; / RTI &gt;


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